CELL CYCLE AND GLUCOCORTICOID RECEPTOR PHOSPHORYLATION

大剂量的糖皮质激素被广泛用于治疗各种疾病 从轻微过敏到白血病和淋巴瘤。他们几乎对所有人都采取行动 细胞。为了最大限度地发挥它们的治疗作用，人们付出了如此多的努力 有效性，并最大限度地减少不必要的副作用。 在细胞周期中，对糖皮质激素的敏感性变化很大， 激素只有在G1后期和S(脱氧核糖核酸)出现时才有效 合成)相。我们的长期目标是阐明基本的机制。 这一变种。在该提案中，中心目标是测试 糖皮质激素受体(GRs)磷酸化改变的假说 通过细胞周期，并至少部分解释了 敏感度。 这一假说基于细胞周期依赖于GR数的证据， 核结合和磷酸化得到了支持--以及手段 来测试它--来自我们发现GR快速、激素依赖的增加 磷酸化，以及我们的鉴定通过磷酸肽图和 小鼠GRs中7个磷酸化位点的测序：4个位于 控制细胞周期的p34cdc2激酶的共同序列。 该提案旨在回答以下具体问题； 1.激素依赖性GR过度磷酸化和/或GR磷酸化 在细胞周期中，部位会发生变化吗？(A)同步WCL2信元(CHO 小鼠GRs过表达的细胞)将被32P标记并处理 用激素检测激素依赖的GRs的磷酸化；(B) 这些GRs中的磷酸化位点将由磷酸肽识别 任何新的多肽的作图和测序。 2.糖皮质激素敏感性和GR数在细胞内是如何变化的 在CHO细胞中循环吗？GRs过度表达的同步CHO细胞将是 用于：(A)测量激素对稳定转染体活性的影响 基因和胸苷激酶；(B)确定GR数量增加是否 G1/S的结合部位是由于细胞内GR蛋白增加所致。 3.GR磷酸化突变体的行为是否与野生型GR不同？ 高表达GRs的CHO细胞在正常磷酸化位点发生突变 将如(2a)所述进行研究。 4.GR磷酸化和糖皮质激素诱导的细胞凋亡 淋巴细胞依赖于细胞周期，并可通过化疗改变 特工？同步的WEHI-7细胞将用于测量(A)GR (B)糖皮质激素诱导的细胞凋亡；(C) 细胞周期调节剂在(A)和(B)中的作用。 我们的结果可能有助于治疗和增进对糖皮质激素的理解 抵抗。

Glucocorticoids at high doses are used widely to treat disorders ranging from minor allergies to leukemias and lymphoma. They act on almost all cells. So much effort has gone into maximizing their therapeutic effectiveness and minimizing unwanted side effects. Sensitivity to glucocorticoids varies strikingly through the cell cycle, the hormones being effective only if present during late G1 and S (DNA synthesis) phases. Our long-term goal is to elucidate the basic mechanisms of this variation. In this proposal, the central objective is to test the hypothesis that phosphorylation of glucocorticoid receptors (GRs) changes through the cell cycle and accounts at least in part for the variation in sensitivity. This hypothesis, based on evidence for cell cycle-dependence of GR number, nuclear binding, and phosphorylation, has gained support -- and the means to test it -- from our finding of a rapid, hormone-dependent increase in GR phosphorylation, and our identification by phosphopeptide mapping and sequencing of seven phosphorylated sites in mouse GRs: four lie in consensus sequences for the p34cdc2 kinases that control the cell cycle. The proposal is designed to answer the following specific questions; 1. Do hormone dependent GR hyperphosphorylation and/or GR phosphorylated sites change through the cell cycle? (a) Synchronized WCL2 cells (CHO cells with overexpressed mouse GRs) will be labeled with 32P and treated with hormone to assay hormone-dependent phosphorylation of the GRs; (b) Phosphorylated sites in these GRs will be identified by phosphopeptide mapping, and sequencing of any new peptides. 2. How do glucocorticoid sensitivity and GR number change through the cell cycle in Cho cells? Synchronized CHO cells with overexpressed GRs will be used to: (a) measure hormone effects on activity of a stably transfected gene and thymidine kinase; (b) determine if the increased number of GR binding sites in G1/S is due to increased GR protein per cell. 3. Do GR phosphorylation mutants behave differently from wild-type GRs? CHO cells with overexpressed GRs mutated at normally phosphorylated sites will be studied as in (2a). 4. Are GR phosphorylation and glucocorticoid-induced apoptosis in lymphocytes cell cycle-dependent, and modifiable by chemotherapeutic agents? Synchronized WEHI-7 cells will be used to measure (a) GR phosphorylation as in la; (b) glucocorticoid-induced apoptosis; (c) effects in (a) and (b) of cell cycle-modifying agents. Our results may benefit therapy and enhance understanding of glucocorticoid resistance.